Studies On Analytical Methods Of Biosensing Based On Rolling Circle Amplification And Nanomaterials

With the continuous development of the analytical science, obtaining the related information of proteins, nucleic acids, enzymes and small molecules in life sciences are increasingly needed with the help of biosensing technology. It is of great significance to fast and accurately obtain the information of these molecules in biomedicine, clinical diagnosis and therapy. In recent years, a seris of new biology techniques have been developed. However, with the constant improvement of the medical diagnosis and the sustained development of the scientific research, techniques with greater sensitivity and specificity for bioanalysis are required. Therefore, it is still a great challenge fo r scientific researchers in analytical chemistry to develop low-cost, simple and rapid strategies with high sensitivity, specificity, accuracy for quantitative or qualitative analysis, which are used to meet the needs of research and practical application. In this thesis, we developed a seris of bioanalysis methods with high sensivity and specifity for enzymes, nucleic acid, proteins and small molecule detection. The feasibility, reliability and accuracy of these methods were investigated through the analysis of real samples. The detailed content was described as follows:Human 8-oxoguanine DNA glycosylase(h OGG1) plays a crucial role in maintaining the genomic integrity of living organisms for its capability of repairing DNA oxidative damage. The expression level of h OGG1 is closely associated with many diseases including various kinds of cancers. In chapter 2, a novel “light-up” sensor based on target-induced formation of 5’ phosphorylated probe and autocatalytic DNAzymegenerated rolling circle amplification has been developed for highly sensitive human 8-oxoguanine DNA glycosylase(h OGG1) activity assay. The approach reaches detection limit as low as 0.001 U/m L for h OGG1 via scarcely increased background signal and dual signal amplification strategy. To the best of our knowledge, it is one of the most sensitive methods for the detection of base excision repair enzyme. Moreover, the approach shows excellent specificity over other nonspecific enzymes would interfere with the assay and holds great promise for application in real sample analysis. Hence, the proposed method provides a highly sensitive, selective, and desirable h OGG1 sensing platform.Single nucleotide polymorphisms(SNPs), which refer to single point mutation in the genome, are the most frequently occurring genetic mutation type. If the mutations are in the region of genetic code, it may cause the change of protein structure or function, thus triggering genetic dieases. Therefore, it has profound implications for the prenatal gene diagnosis to develop strategies with high sensitivity and high specificity used in the detection of single nucleotide polymorphisms. In chapter 3, we established a method with high sensitivity and high specificity for single nucleotide polymorphisms detection based on the highly specific recognition capability of Ecoli DNA ligase to single mismatch and the combination of rolling circle amplification with Mg2+-DNAzyme, which has been used in the above chapter. The padlock probe was able to be ligated into circular probe when the target DNA matched with padlock probe perfectly,and then triggered cascadic signal amplification reaction. However, when there was a mismatch in the hybridized DNA complex, the padlock probe cannot be cyclized by Ecoli DNA ligase and the cascadic sign al amplification could not be conducted.Thus, no desirable fluorescence signal was obtained. The sensor showed good sensing performance with good linearity ranging from 0.01 n M to 1 n M and a detection limit of 2.6 p M. Moreover, owning to the high fidelity in distinguishing the mismatched bases at the ligation site of DNA ligase, positive detection of mutant targets was able to be achieved when the wild-type and the mutant type DNA were mixed in various ratio.Endonuclease IV(Endo IV), as one of DNA repairing enzymes, plays a crucial role in repairing damaged DNA comprising of abasic sites to maintain the genomic integrity. In chapter 4,the cleaving capability of Endo IV to apurinic/apyrimidinic sites(AP) in single-stranded DNA(ss DNA) was demonstrated. It was found that Endo IV has considerably high cleaving activity to AP sites in ss DNA compared with that in double-stranded DNA(ds DNA). The unique feature of Endo IV in cleaving AP sites in ss DNA was further applied to construct a novel dual signal amplifi ed sensing system for highly sensitive enzyme and protein detection by combination of Exonuclease III(Exo III)-aided cyclic amplification reaction and rolling circle replication(RCR) technique, which showed good sensing performance with a detection limit of 0.008 U/m L for Endo IV and 2.5 p M for streptavidin. In addition, the developed method had considerably high specificity for Endo IV and strepavidin over other potential interferences. The developed strategy indeed provides a novel platform for proteins and enzymes assay and may find a broad spectrum of applications in bioanalysis, diseases diagnosis, and drug development.Acetylcholinesterase(ACh E) is a critical enzyme in animal’s central nervous system and has pivotal functions in Alzheimer’s disease, inflammatory processes and nerve agent poisoning. ACh E maintains the level of the neurotransmitter by hydrolysis of acetylcholine(ACh) to choline. However, inhibition of ACh E activity by its inhibitors, allows ACh to remain active and accumulate throughout the body, which may lead to fatal consequences. Therefore, it is of great importance to assay the activity of ACh E and screen its potential inhibitors. In chapter 5,We have developed a convenient, highly sensitive and selective flu orescence assay for acetylcholinesterase activity(ACh E) and inhibitor detection by using the poly(thymine)-templated fluorescent copper nanoparticles(poly T-templated Cu NPs) as fluorescence indicator. In this assay, acetylthiocholine chloride(ATCh) was introduced as the substrate of ACh E, and it could be catalytically hydrolyzed by ACh E to produce thiocholine whose thiol was able to effectively quench fluorescent poly T-templated Cu NPs via coordination with Cu NPs. The sensor exhibited excellent analytical performance for ACh E in the concentration of 0 m U/m L to 5 m U/m L with a detection limit of 0.026 m U/m L and high specificity against other nonspecific proteins. In addition, organophosphorus pesticide paraoxon was employed as the model inhibitor to inhibit the hydrolysis of ATCh and recover the fluorescence of the poly T-templated Cu NPs. The IC50 of paraoxon was estimated to be 84 pg/m L. Furthermore, the sensing strategy can be used to detect spiked paraoxon in real samples. The present work may provide an attractive platform for the field of enzyme sensors in biochemical and clinical applications.Fluorescent nanoparticles receive increasing interest nowadays for their potential applications in labeling, sensing, imaging and biomedical applications. In chapter 6, we reported the synthesis of intrinsic fluorescent polydopamine(PDA) nanoparticles using Mn O2 as oxidant. In the presence of Mn O2, dopamine was quickly oxidized into its quinone derivative, autopolymerized into fluorescent PDA nanoparticles. Using the fluorescent PDA nanoparticles as a fluorescence signal indicator, we further established a cost-effective sensor for rapid, sensitive and selective GSH assay based on the redox reaction between Mn O2 and GSH, and the key role of Mn O2 in the formation of the fluorescent PDA nanoparticles. GSH has the capability of reducing Mn O2 into Mn2+, which inhibited the formation of the fluorescent PDA nanoparticles for the asence of the oxidizing Mn O2. Thus, the concentration of GSH was directly related to the decreased signal intensity of the fluorescent PDA nanoparticles. The sensor showed good sensing performance for GSH detection with high sensivity and desirable selectivity over other potent ial interfering species. Additionally, the sensor exhibited excellent practical applications for GSH detection in human whole blood samples, which presents potential applications in biological detection and clinical diagnosis.